The human hematopoietic system is populated by cells of several different lineages. These "blood cells" may appear in bone marrow, the thymus, lymphatic tissue(s) and in peripheral blood. Within any specific lineage, there are a number of maturational stages. In most instances, the more immature developmental stages occur within bone marrow while the more mature and final stages of development occur in peripheral blood.
There are two major lineages: The myeloid lineage which matures into red blood cells, granulocyte, monocytes and megakaryocytes; and the lymphoid lineage which matures into B lymphocytes and T lymphocytes. Within each lineage and between each lineage, antigens are expressed differentially on the surface and in the cytoplasm of the cells in a given lineage. The expression of one or more antigens and/or the intensity of expression can be used to distinguish between maturational stages within a lineage and between lineages.
Assignment of cell to lineage and to a maturational stage within a cell lineage indicates lineage commitment. There are cells, however, which are uncommitted to any lineage (i.e., "progenitor" cells) and which, therefore, retain the ability to differentiate into each lineage. These undifferentiated, pluripotent progenitor cells will hereinafter be referred to as the "stem cells."
All of mammalian hematopoietic cells can, in theory, be derived from a single stem cell. In vivo, the stem cell is able to self-renew, so as to maintain a continuous source of pluripotent cells. In addition, when subject to particular environments and/or factors, the stem cells may differentiate to yield dedicated progenitor cells, which in turn may serve as the ancestor cells to a limited number of blood cell types. These ancestor cells will go through a number of stages before ultimately yielding mature cells.
The benefit of obtaining a pure population of stem cells is most readily recognized in the field of gene therapy. Gene therapy seeks to replace or repopulate the cells of the hematopoietic system which contain a defective gene with cells that do not contain the defective gene but instead contain a "normal" gene. Using conventional recombinant DNA techniques, a "normal" gene is isolated, placed into a viral vector, and the viral vector is transfected into a cell capable of expressing the product coded for by the gene. The cell then must be introduced into the patient. If the "normal" gene product is produced, the patient is "cured" of the condition. The difficulty is that the transformed cells must be capable of continual regeneration as well as growth and differentiation.
Although stem cells are potentially optimal "hosts" for transformation, substantial problems have been encountered in (a) identifying the antigenic markers unique to stem cells, (b) isolating homogenous populations comprising substantial numbers of non-lineage committed, pluripotent stem cells and (c) maintaining and, possibly, expanding populations of human stem cells.
However, a number of research groups have recently reported the isolation of populations of mammalian bone marrow cell populations which are enriched to a greater or lesser extent in pluripotent stem cells. For example, C. Verfaillie et al., J. Exp. Med., 172, 509 (1990) reported that a two-step purification of low density human bone marrow cells by negative immunomagnetic selection and positive dual-color fluorescence activated cell sorting (FACS) yielded a Lin.sup.- /CD34.sup.+ /HLA-DR.sup.- cell fraction that was 420-fold enriched in pluripotent stem cells capable of initiating long-term bone marrow cultures (LTBMC) over unmanipulated bone marrow mononucleocytes (BMMNC) obtained after Ficoll-Hypaque separation. This group reported that the combination of positive selection for small blast-like cells that are CD34 antigen positive but HLA-DR antigen negative, combined with a more extensive negative selection to deplete the population of CD2, CD19 and CD71, results in an about two- to three-fold greater enrichment in pluripotent stem cells over that Previously reported.
The development of cell culture media and conditions that will maintain stem cells in vitro for the extended periods of time required for the procedures involved in gene therapy, identification of growth factors, thorough characterization of cell morphologies and the like, has presented a unique set of obstacles. To date, successful in vitro stem cell cultures have depended on the ability of the laboratory worker to mimic the conditions which are believed to be responsible for maintaining stem cells in vivo.
For example, hematopoiesis occurs within highly dense cellular niches within the bone marrow in the adult and in similar niches within the fetal yolk sac and liver. Within these niches, stem cell differentiation is regulated, in part, through interactions with local mesenchymal cells or stromal cells. Mammalian hematopoiesis has been studied in vitro through the use of various long-term marrow culture systems. T. M. Dexter et al., in J. Cell Physiol., 91, 335 (1977) described a murine system from which spleen colony-forming units (CFU-S) and granulocyte/macrophage colony forming units (CFU-GM) could be detected for several months, with erythroid and megakaryocytic precursors appearing for a more limited time. Maintenance of these cultures was dependent on the formation of an adherent stromal cell layer composed of endothelial cells, adipocytes, reticular cells, and macrophages. These methods were soon adapted for the study of human bone marrow. Human long-term culture system were reported to generate assayable hematopoietic progenitor cells for 8 or 9 weeks, and, later, for up to 20 weeks (See, S. Gartner, et al., PNAS USA, 77, 4756 (1980); F. T. Slovick et al., Exp. Hematol., 12, 327 (1984). Such cultures were also reliant on the preestablishment of a stromal cell layer which must frequently be reinoculated with large, heterogeneous populations of marrow cells. Hematopoietic stem cells have been shown to home and adhere to this adherent cell multilayer before generating and releasing more committed progenitor cells (M. Y. Gordon et al., J. Cell Physiol., 130, 150 (1987)).
Stromal cells are believed to provide not only a physical matrix on which stem cells reside, but also to produce membrane-contact signals and/or hematopoietic growth factors necessary for stem cell proliferation and differentiation. This heterogenous mixture of cells comprising the adherent cell layer presents an inherently complex system from which the isolation of discrete variables affecting stem cell growth has proven difficult. Furthermore, growth of stem cells on a stromal layer makes it difficult to recover the hematopoietic cells or their progeny efficiently.
C. M. Verfaillie, in Blood, 79, 2821 (1992) and P. McGlave et al., in U.S. patent application Ser. No. 07/867,814, filed Apr. 3, 1992, demonstrated that primitive, lineage-non-committed CD34.sup.+ /HLA-DR.sup.- cells can differentiate and can be maintained when cocultured with stromal layers but separated from the stromal layers by a 0.4 .mu.m microporous membrane ("stroma non-contact" culture). In U.S. patent application Ser. No. 08/032,670, filed Mar. 17, 1993, P. McGlave and C. Verfaillie demonstrated that Lin.sup.- /CD34.sup.+ /HLA-DR.sup.- cells can differentiate and proliferate when they are cultured without a stromal layer ("stroma free culture") but are supplemented daily by media conditioned by normal allogeneic bone marrow stromal layers. These studies suggest that soluble factors derived from the bone marrow stromal layers are capable of inducing differentiation of primitive human hematopoietic cells and can conserve at least a fraction of more primitive progenitors.
One role of the stromal cells in stroma-dependent cultures may be to provide a combination of cytokines that promote differentiation and proliferation of primitive hematopoietic progenitors. See, for example, E. L. W. Kittler et al., Blood, 12 3168 (1992) and C. J. Eaves et al., Blood, 78, 110 (1991). Long-term cultures can indeed be established from primitive hematopoietic progenitors in the absence of an adherent stromal layer when defined cytokines are repeatedly added. See, for example, J. Brandt et al., J. Clin. Invest., 86, 932 (1990); L. W. M. M. Terstappenet ed., Blood, 77, 1218 (1991); G. Migliascio et al., Blood, 79, 2620 (1992) and D. N. Haylock et al., Blood, 80, 1405 (1992). Cytokines thought to be important in the induction of differentiation and/or proliferation of primitive hematopoietic progenitors are listed on Table 1, below.
TABLE 1 ______________________________________ Cytokine Reference ______________________________________ rhu-G-CSF* K. Ikebuchi et al., PNAS USA, 85, 3445 (1988) rhuIL-1, rhuIL-6, J. Brandt et al., J. Clin. Invest., 82, 1017 (1988); rhuIL-3 A.G. Leary et. al., Bood, 71, 1759 (1988) rhuIL-11 S.R. Paul et al., PNAS USA, 87, 7512 (1990); K. Tsuji et al., PNAS USA, 87, 7512 (1990) LIF (leukemia F.A. Fletcher et. al., Blood, 76, 1098 (1990) inhibitory factor) SCF (stem cell J. Brandt et al., Blood, 79, 634 (1992); K.M. factor) Zsebo et al., Cell, 63, 195 (1990) bFGF S. Huang et al., Nature, 360, 745 (1992) rhuGM-CSF J. Brandt et al., J. Clin. Invest., 86, 932 ______________________________________ (1990) *rhu = recombinant human
A variety of culture systems have been described in which cells are cultured with combinations of these growth promoting cytokines in the absence of stroma-conditioned medium or stromal feeders (ex vivo cultures). For example, Haylock et al. reported that culture of mobilized peripheral blood CD34.sup.+ cells with IL-1, IL-3, IL-6, G-CSF, GM-CSF and SCF increase CFU-GM by a mean of 66-fold. Haylock et al., Blood, 80, 1405 (1992). Additionally, Brugger et al. reported a 190-fold expansion of clonogenic progenitors following culture of mobilized peripheral blood CD34.sup.+ cells with SCF, erythropoietin, IL-1, IL-3 and IL-6. Brugger et al., Blood, 81, 2579 (1993). However, these cultures usually do not allow expansion or even maintenance of more primitive progenitors.
The role of cytokines in the hematopoiesis occurring in long-term bone marrow cultures remains uncertain, and the factors that regulate both self-replication and the initial differentiation process of primitive uncommitted hematopoietic progenitors are still largely unknown. Therefore, there is a continuing need to characterize and evaluate factors produced by the stromal cells in long-term cultures, and to uncover and elucidate the mechanism underlying the self-replication and initial differentiation of the human hematopoietic stem cell. Characterization of such stroma-derived factor(s) may have important clinical applications, such as ex vivo stem cell expansion for use in cancer treatment and gene therapy.